Processing and storage

Serum, plasma, and whole blood are complex matrices. We tend to forget that after collection and subsequent processing, the samples are far from static. As the samples are allowed to sit, carbon dioxide is lost and the pH changes. Enzymes present may lose activity or gain activity. Chemical interactions between various endogenous and exogenous chemicals occur between and amongst each other. Recognizing the potential for these and understanding how they impact the stability of a given drug is important (29,30).

One of the best examples comes from toxicology when cocaine needs to be measured in blood. The metabolism of this drug by circulating cholinesterases continues unless fluoride is used to inhibit the process. Similarly, interactions between two drugs administered may continue after sample collection. For example, it is not unusual for several antibiotics to be used sequentially to treat some infections. One practice involves the use of both an aminoglycoside and a beta-lactam antibiotic. The drugs are not co-administered in the same infusion because it is known that beta-lactams will inactivate aminoglycosides rendering the aminoglycoside ineffective against the microorganism for which it is intended and unrecognizable by the antibodies in the immunoassays used to quantify serum concentrations. The rate at which inactivation occurs depends upon the aminoglycoside involved, the beta-lactam antibiotic involved, as well as the time and temperature of exposure (31). Although administered sequentially to avoid this in vivo, both drugs may be present in the circulation and subsequently in the collection tube when samples are collected. Consequently, the amount of measurable aminogly-coside will decline over time leading to lower and lower apparent concentrations. It is therefore advisable to process, separate, and analyze the samples immediately or to freeze the samples if testing is delayed (31).

Methotrexate has long been considered to be unstable. Limelette et al. (32) investigated this question by spiking methotrexate into whole blood collected from healthy donors. The blood was collected on citrate-phosphorus-dextrose. Using an HLPC-based method, they compared whole blood and plasma methotrexate concentrations from these samples over time and various storage conditions. The whole blood stability study was performed using a pool, which was then divided into three sub-pools. The sub-pools were stored at room temperature exposed to light, at room temperature protected from light, and at 4°C protected from light. Aliquots were tested immediately after preparation and at intervals up to 144 h. Visual inspection of the graphic display of the resulting data shows similar changes in the methotrexate concentrations regardless of the storage conditions from initial preparation until 48 h later. Concentrations declined slightly, but the deviation from baseline was less than 10%. After 48 h, there was little change in the levels observed for the light-protected pool stored at 4°C. Interestingly, of the two pools retained at room temperature, the one protected from light showed the greatest loss of methotrexate with changes of more than 20% occurring by 100 h.

When the investigators tested three plasma pools under the same conditions, they found that the drug concentrations also steadily declined by approximately 17% over the first 3 days of testing before leveling off. Little change was observed over an additional 7 days. The study has the following limitations: only one concentration of methotrexate was tested, the samples were prepared pools not patient samples, and the blood used to prepare the pools did not simulate typical patient samples in that it contained citrate-phosphorus-dextrose. Because the use of methotrexate extends beyond its use as a chemotherapeutic agent for leukemia and other cancers and monitoring is widely performed, it would thus be of great use to clinical laboratories to simplify the collection, processing, and storage of specimens.

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